Comparing Imaging Depth of Intravital Lung Imaging Using Perfluorocarbon‐Based Liquid Ventilation With Tissue Clearing for Deep‐Tissue Imaging

Intravital lung imaging has been employed to study physiological and pathophysiological processes related to nanoparticle deposition in the alveolar lung, particularly in the context of air pollution and drug delivery. However, optical imaging depth is limited, often attributed to the refractive index (RI) mismatch at the alveolar air‐tissue interface. To investigate this, we evaluated two complementary strategies. First, we demonstrated that eliminating the RI mismatch via partial liquid ventilation with oxygenated perfluorocarbon (PFC) did not enhance the imaging depth. A second approach, utilizing ex vivo optical tissue clearing (with RI matching), was only successful in improving imaging penetration depth if it included removal of scattering lipids such as pulmonary surfactant. Nevertheless, partial liquid ventilation with PFC in vivo enabled the homogeneous delivery of nanoparticles to the alveoli, allowing real‐time observation of their interactions with lung epithelium. This finding opens new avenues for studying inhaled particulates and optimizing inhalation‐based drug delivery.